Protein antibiotic finds novel way of sn(e)aking across the membrane

Research into the molecular antics of bacterial protein antibiotics has revealed a novel mechanism for delivering a signal into cells.

The unfolded N-terminal region of the colicin lodges itself in the pore of an OmpF subunit (adapted from (2)). Only a single subunit of the OmpF trimer is shown (Click to enlarge)

Colin Kleanthous and his group, together with colleagues in the Chemistry department in Oxford and at Birkbeck College, describe the snaking mechanism in their recent paper in Science (1).

The unusual process by which these protein antibiotics initiate entry into cells could be relevant to other biological systems where proteins have to get across membranes. The work will also help studies exploring whether these antibiotics, which target closely related bacteria, have clinical potential.

Colicins are part of a large family of antibacterial proteins that target the gut bacterium Escherichia coli. Similar antibiotics are produced by many Gram-negative bacteria, including many pathogens, and are used to target neighbouring organisms competing for the same resources.

They bind to specific receptor proteins on the cell surface and then assemble a complex nanomachine or ‘translocon’ that links the outside of the cell to the inside. Once these connections are made, the colicin is able to transit into the cell by a mechanism that is still being elucidated.

OmpF is an outer membrane pore protein which is recruited to become part of the translocon. Binding of OmpF allows the colicin to contact TolB, a periplasmic protein, which in turn promotes interaction with an inner membrane protein, TolA. As this happens, there is an energy transduction step which catalyses the entry of colicin into the cell. Once inside, the colicin delivers its toxic payload.

An unfolded region in the colicin was known to recruit OmpF in order to pass a signal, in the form of a peptide, across the bacterial outer membrane to TolB. But until now, the details of how the colicin exploited OmpF were unknown. The work from Professor Kleanthous and colleagues now shed light on the process, revealing a surprising ‘snaking’ mechanism.

The conclusions described in the Science paper required important technical developments in a number of areas.

Mass spectrometry of the intact ColE9 translocon. The colicin, ColE9 (brown), contains an intrinsically unstructured translocation domain shown as a dotted line, which passes through the trimeric porin OmpF (green). The translocon also comprises the immunity protein Im9 (yellow) bound to the C-terminal domain of ColE9 and the membrane receptor protein BtuB (blue). An engineered (disulphide) crosslink (red sphere) between ColE9 and histidine-tagged TolB (purple) helps stabilise the translocon so it can be purified. Coloured inserts give assignments for species observed in the spectrum: orange hexagon, the intact complex that includes a single molecule of lipopolysaccharide; red circle, translocon from which BtuB and lipopolysaccharide have dissociated; blue square, dissociated BtuB

One of the major advances was in mass spectrometry, in collaboration with Professor Carol Robinson and Dr Jonathan Hopper in the Chemistry department. ‘The work we’ve done together is really pushing the boundaries in terms of membrane protein mass spectrometry’, says Professor Kleanthous. ‘The mass spec result was amazing - it had never been done before.’

The researchers suspected that the unfolded region of colicin threaded through two of the three holes of the 3 subunit OmpF molecule. To investigate this, they designed a mass spectrometry approach that allowed them to measure the mass (weigh) OmpF in the gas phase with or without a peptide that originated from the colicin’s unfolded region and known to lodge in these holes (2).

The group also used channel measurements, in collaboration with Professor Hagen Bayley and Dr David Rodriguez-Larrea in Chemistry, to check the hole occupancy of OmpF.

‘The mass spec approach showed that we could measure the mass of the peptide inside the holes’, explains Professor Kleanthous. ‘The peptide is only around 1% of the total mass, but we can detect this change in mass because the resolution of the technique is so good.’

Armed with these approaches, the group set out to explore what was happening in the translocon. But to do this they had to find a way of stabilising the complex since this is a transient species formed just before the colicin passes into the cell. They achieved this by engineering a mutation in the colicin so that it remained covalently linked to the TolB protein once it had threaded through OmpF.

This allowed them to capture the colicin translocon, purify it and embark on a detailed biophysical and structural analysis. Both the mass spectrometry and the electrophysiology experiments demonstrated that in forming its translocon the colicin had snaked in and out of two subunits of the OmpF protein, leaving one subunit unoccupied.

‘We found that colicin is tethered to two holes in a three-hole protein’, explains Professor Kleanthous. ‘The surprise is that the colicin not only goes into the cell by one of the holes of OmpF but also comes back out again through a second hole.’

Structural representation of the ColE9 translocon depicting how the colicin twice traverses the outer membrane to capture TolB in the periplasm in a fixed orientation. The colicin induces TolB to interact with TolA, which is part of the energy transducing TolQRA system of the inner membrane (Click to enlarge)

The functional significance of tethering to two OmpF subunits became apparent when the group carried out electron microscopy studies of part of the translocon in collaboration with Professor Helen Saibil and Dr Natalya Lukoyanova at Birkbeck College. They saw that the tethered colicin enables TolB to be held rigidly, even though it sits on the other side of the membrane, presenting it in a fixed orientation to the inner membrane protein TolA, the next protein in the relay.

Professor Kleanthous comments that the mechanism explains how disordered proteins can burrow their way through narrow pores, as well as pass a charged signal into a cell.

‘This is a mechanism by which the protein essentially hides its charges within the charged holes of OmpF so that it can pass a peptide signal across the hydrophobic membrane into the cell.’

The research represents the culmination of work which Professor Kleanthous and his group, in particular his long-standing postdoc Dr Nick Housden who developed the translocon trapping strategy, started when they were in York. The work has come to fruition with the move to Oxford, enabling new collaborators to come on board.

Now that the group have started to piece together the molecular interactions between different components of the translocon, they are keen to fill in more of the details.

‘We want to find out exactly how the colicin sneaks its way in and out of OmpF and to see if this mechanism occurs in colicins that target pathogenic bacteria’, says Professor Kleanthous. ‘We also need to figure out how the translocon catalyses internalisation of the colicin. We’ll be focusing on all the components of the translocon, ultimately trying to assemble them in a reconstituted system in vitro.’